Hiv-like particles and the use thereof

ABSTRACT

Disclosed are alphavirus-based expression vectors expressing env,gag,pro, and/or pol genes of HIV-1, their transcripts, and transformed host cells. The present invention describes the expression of the Gag,Env,Pol and/or Pro Proteins using the above expression vectors, and HIV-like particles(HTVLPs) composed of the above recombinant proteins. The virus-like particles (VLPs) of the present invention, as the matuer and infective particles, are very useful as an antigen for a diagnostic kit for HIV infection as well as for vaccine composition for prevention of HIV infection.

TECHNICAL FIELD

[0001] The present invention relates to an expression vector whichexpresses Gag protein of HIV, an expression vector which expresses Envprotein of HIV, an expression vector which expresses both Gag and Envproteins simultaneously, an expression vector which expresses Gagpolpolyprotein of HIV and an expression vector which expresses both Gagpolpolyprotein and Env protein simultaneously.

BACKGROUND ART

[0002] Human Immunodeficiency Virus (HIV) is known as the causativeagent of Acquired Immunodeficiency Syndrome (AIDS). Especially, HIV-1and HIV-2 belongs to the subgroup Lentiviridae of retroviruses, whichcarry a single-stranded RNA. Once in a susceptible host cell, the RNAgenome is reverse-translated by viral reverse transcriptase to generatedouble-stranded DNA, which becomes integrated into the host'schromosome, resulting in provirus.

[0003] In order to better understand the background of the presentinvention, with reference to FIG. 1, there is given the structure of theHIV-1 genome 9.2 kb in size. The HIV-1 genome possesses structural genesconsisting of gag, pol and env, and accessory genes including tat, rev,nef, vif, vpu and vpx.

[0004] A HIV Gag polyprotein precursor, Pr55gag, is expressed fromfull-length gag-pol RNA with a size of 55 kDa. A HIV Gag precursor istransported to the inner surface of the cell plasma membrane by aN-terminal myristate moiety, where directing the assembly of virions.Maturation of virions requires the proteolytic cleavage of the Pr55gagby protease (Pro) encoded by the pol gene of HIV into matrix (MA, p17),capsid (CA, p24), nucleocapsid (NA, p9) and p6 proteins (Gheysen D. etal, Cell, 59, 103-112 (1989); Bray, M. et al. Proc. Natl. Acad. Sci. USA91, 1256-1260 (1994); Cann, A. J., and J. Karn, AIDS 3(Suppl.1), S19-S34(1989); Ratner, L., W. et al., Nature 313, 277-284 (1985); Wain-Hobson,S. et al., Cell 40, 9-17 (1985)).

[0005] The envelope glycoprotein (Env) precursor, gp160, is expressedfrom the spliced RNA. In virus-infected cells, the precursor is cleavedby protease from the host or the virus into gp120 subunit at theN-terminal side and gp41 subunit at the C-terminal side, which areproperly targeted to a plasma membrane with anchorage on the externalsurface and insertion into the cell membrane, respectively. In themature virion, gp120 and gp41 subunits form a heterodimer in which thetwo subunits are non-covalently associated, which is known to bedirectly responsible for the infectivity, cellular tropism andcytopathogenicity of HIV (Robey, E. and Axel, R., Cell 60, 697-700(1990)). Because of the close relation to immunogenicity, the Envprotein of HIV is a target for the development of the HIV vaccines.

[0006] The protease (Pro) is produced from self-cleavage of Pr160gag-pol precursor, translated by ribosomal frame shift at 3′ end of gagmRNA region while translating gag-pol mRNA (Jacks, T. and Varmus, H. E.Science 230, 1237-1242 (1985); Jacks, T. et al, Nature 331, 280-283(1988); Sonigo, p. et al, Cell 45, 375-385 (1986)). The proteaseparticipates in the processing of the Pr160gag-pol polyprotein and envprotein, resulting in mature infectious virions (Kohl, N. et al., Proc.Natl. Acad. Sci. USA 85, 4686-4690 (1988)).

[0007] To date, suggested AIDS vaccines include inactivated vaccines,attenuated vaccines, recombinant live vaccines, virus-like particlevaccines, and subunit vaccines.

[0008] The inactivated whole virus vaccines are prepared by chemicallyor physically treating isolated viral particles to destroy theirinfectivity. This kind of vaccine is easy to prepare and is capable ofusing most epitopes, in contrast, it may cause diseases when the virusis not completely inactivated, on the other hand, the strong treatmentfor the complete inactivation may cause destruction of many epitopes.Although there are some successful reports on the inactivated SIVvaccines (Scott E. J. Nature 253, 393 (1991); Le Grand R. et al., Nature355, 684 (1992); Crange M. P. et al., Nature 355, 685-686 (1992); ArthurL. O. et al., J Virol 69, 3117-3124 (1195); Neidrig M. et al., Vaccine11, 67-74 (1993)), it is not considered useful as a safe and effectiveAIDS vaccine because suspicion has been raised that the successfulimmunization resulted from action by xeno-antigen which the cultivatedhost cells harbor.

[0009] The live attenuated vaccines are prepared by partial deletion inthe HIV genome, such as deletion of nef gene or nef and vpr genes(Descrosiers R. C. AIDS Res Hum Retroviruses 8, 411-421 (1992); Gibbs J.S. et al., AIDS Res Hum Retroviruses 10, 607-616 (1994); Cranage M. P.et al., Virology 229, 143-154 (1997); Wyand M. S. et al. Nature Med 3,32-36 (1997)). However, such vaccines can regain the deleted genes invaccinated animals, allowing them to be infectious. Accordingly, thelive attenuated vaccines. may not be completely safe because they cancause AIDS in vaccinated individuals (or animals).

[0010] The live recombinant vaccines are vectors in which nonessentialgenes of non-pathogenic viruses are replaced with genes coding HIVimmunogens. There was disclosed an expression of gag, pol or env geneand an expression of an immunogenic region including V3 loop of HIV as achimeric form in vaccinia virus, poxvirus, canarypox virus, avipox virusand poliovirus, or influenza virus (Tartaglia J. et al., Virology, 188,217-232 (1992); Abimiku A. et al., Nature Med, 1, 321-329 (1995); NatukR. J. et al., AIDS Hum Retroviruses 9, 395-404 (1993); Li S. et al., JVirol, 67, 6659-6666 (1993); Muster T. et al., J Virol 69, 6678-6686(1995)). As this technology, in general, uses a part of the protein as avaccine material, it cannot provide an efficient immunogen. In addition,the live recombinant vectors are unstable (Morrow. C. D. et al., AIDSRes Hum Retroviruses, 10, S61-S66 (1994); Anderson M. J. et al., AIDSRes Hum Retroviruses, 13, 53-62 (1997)).

[0011] The subunit vaccines are subunits of proteins encoded by HIV,which are naturally isolated or expressed by recombinant DNA technology.These vaccines can be easily manufactured, but they have a differentconformation from original proteins existing in viral particles.Recombinant Env glycoproteins, gp120 and gp160, were expressed usingmammalian and insect cell expression systems (Lasky, L. A., et al.,Science, 249, 932-935 (1986); Barr, P. J. et al., Vaccine 5, 90-101(1987); Hu, S-L. et al., J Virol 61, 3617-3620 (1987); Rusche, J. R. etal., Proc. Natl. Acad. Sci., 84, 6924-6928 (1987); Berman, P. W., et al.J Virol 63, 3489-3498 (1989); Ivey-Hoyle, M., and Rosenberg, M., Mol.Cell. Biol., 10, 6152-6159 (1990); Lasky, L. A. et al., Cell, 50,975-985 (1987)). Upon being injected into chimpanzees, gp120subunitvaccine expressed in the CHO cells could not confer protection against aviral challenge experiment (Berman, P. W. et al., Proc. Natl. Acad.Sci., 85, 5200-5204 (1988)).

[0012] Generally, vaccines of viral particle form are more immunogenicand stable than those of subunit form. There was recently developed anew technology in which the non-infectious virus-like particles (VLPs)that do not carry genetic materials were used as vaccines. For example,mutation in packaging genes can provide a VLP without an RNA genome, anda VLP containing unprocessed gag protein can be prepared usingrecombinant baculovirus or vaccinia virus (Gorelink R. J. et al., JVirol 64, 3207-3211 (1990); Gheysen D. E. et al., Cell 59,103-112(1989)). It was also reported that virus-like particles wereproduced from the cells co-transfected with two recombinant vacciniaviruses containing gag-pol gene and env gene, respectively (Haffar O. K.et al., Virology, 183, 487-495(1991)). Such pseudo-particles wereeffective for production of neutralizing antibodies, env-CD4 protectingantibodies, and syncytium inhibition, but they have the drawbacks intheir not being mature virus particles and lack of infectivity.

[0013] As other forms of virus-like particles containingimmunodeterminant of HIV, there were described hybrid particlesincluding poliovirus capsovector, hepatitis B virus (HBV) coreparticles, HBsAg particles and yeast retrotransposon virus-likeparticles. But, these hybrid particles contain only the partial regionof the immunodeterminant of HIV, resulting in non-effective vaccines(Grene E. et al., AIDS Res Hum Retroviruses, 13, 41-51 (1997);Schlienger K., et al. J Virol 66, 2570-2576 (1991); Adams S. E. et al.,Nature 329, 68-70 (1987); Layton G. T. et al., J Immunol, 151, 1097-1107(1993); Eckart L. et al., J Gen Virol 77, 2001-2008 (1996)).

[0014] The inventors of the present invention attempted to develop anovel AIDS vaccine with mature virus-like particles using an alphavirusvector system. The inventors believe the virus-like particles willprovide the way to prevent AIDS.

DISCLOSURE OF THE INVENTION

[0015] It is an object of the present invention to provide an expressionvector expressing Gag protein, an expression vector expressing Envprotein of HIV, an expression vector simultaneously expressing Gag andEnv proteins of HIV, an expression vector expressing Gagpol polyproteinof m, and an expression vector simultaneously expressing Gagpolpolyprotein and Env protein of HIV

[0016] It is another object of the present invention to providetransformants transformed with the above expression vector.

[0017] It is a further object of the present invention to provideHIV-like particles HIVLPs), a preparation method thereof, and usethereof, in which the above expression vector or its RNA transcript isintroduced into animal cells.

[0018] It is still a further object of the present invention to providea method of detecting HIV using a mature HIV-like particle and itsapplication in diagnosis of HIV infections.

[0019] It is a yet further object of the present invention to provide avaccine composition comprising the mature HIV-like particles, and alsoprovide an AIDS vaccine composition comprising the expression vector orits RNA transcript.

[0020] It is a still further object of the present invention to providea method of detecting the existence of antibodies specific for HIVantigens in a test sample, comprising the steps of: (a) collecting atest sample which may contain specific antibodies against HIV; (b)reacting the test sample with the mature HIV-like particle preparedaccording to the method of claim 21 under a condition which allows theantigen-antibody immune complex formation; and (c) detectingantigen-antibody complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a brief view showing a genome structure of HIV-1;

[0022]FIG. 2 is a restriction map showing structures of expressionvectors pSFV and pSFV-helper;

[0023]FIG. 3 is a schematic diagram showing a process for constructionof an expression vector pSFV/gag;

[0024]FIG. 4 is a schematic diagram showing a process for constructionof an expression vector pSFV/gagpro;

[0025]FIG. 5 is a photograph showing western blotting of HIV-likeparticles with an AIDS patient serum, where the HIV-like particles wereproduced in BHK-21 cells transfected with pSFV-helper and pSFV/gag, orpSFV-helper and pSFV/gagpro, and RNA transcript of pSFV/gagpro;

[0026]FIG. 6 is a photograph showing western blotting of HIV-likeparticles with an AIDS patient serum, where the HIV-like particles wereproduced in infected BHK-21 cells with in vitro activated HIV-likeparticles, prepared from BHK-21 cells transfected with pSFV-helper andpSFV/gag, or pSFV-helper and pSFV/gagpro, and RNA transcript ofpSFV/gagpro;

[0027]FIG. 7 is a photograph showing a result of immunocytochemistry forGag protein in BHK-21 cells transfected with pSFV/gag or pSFV/gagprovector, staining with anti-p24 polyclonal antibody;

[0028]FIG. 8 is a photograph showing an electro-microscopically observedresult for negative-staining of VLPs, isolated from supernatant from acultivated medium of BHK-21 cells transfected with pSFV/gag vector (A)or pSFV/gagpro RNA transcript (B)(X140,000);

[0029]FIG. 9 is a schematic diagram showing a process for constructionof a pSFV/env expression vector;

[0030]FIG. 10 is a photograph showing a result of immunocytochemistryfor Gag protein in BHK-21 cells transfected with pSFV/env RNA transcriptand pSFV-helper RNA transcript, staining with anti-gp160 monoclonalantibody;

[0031]FIG. 11 is a photograph showing western blotting of cell lysatesof BHK-21, infected with in vitro activated defective SFV particles,with an AIDS patient serum, where the defective SFV particles preparedfrom BHK-21 cells transfected with pSFV/env RNA transcript andpSFV-helper RNA transcript;

[0032]FIG. 12 is a photograph showing a result of immunocytochemistryfor Gag and Env proteins of HIV-1 in BHK-21 cells co-transfected withpSFV/gag and pSFV/env RNA transcript, staining with anti-gp160monoclonal antibody (B) or with an AIDS patient serum (C);

[0033]FIG. 13 is a photograph showing agarose gel electrophoresispattern for RT-PCR and PCR products of RNA genome packaged in VLPs,which produced in BHK-21 cells transfected with pSFV/gag RNA transcript,or with pSFV/gag and pSFV/env RNA transcript;

[0034]FIG. 14 is a photograph for western blotting showing production ofVLPs composed of Gag and Env in infected BHK-21 cells with in vitroactivated defective SFV particles, prepared in BHK-21 cellsco-transfected with pSFV-helper and pSFV/gag RNA transcript, or withpSFV-helper and pSFV/env RNA transcript;

[0035]FIG. 15 is a schematic diagram showing a process for constructionof an expression vector pSFV/pro;

[0036]FIG. 16 is a schematic diagram showing a process for constructionof an expression vector pSFV/env-gag;

[0037]FIG. 17 is a schematic diagram showing a process for constructionof an expression vector pSFV/env-gag-pro;

[0038]FIG. 18 is a photograph showing western blotting of cell lysatesor VLPs from BHK-21, transfected with pSFV/env-gag RNA transcript, withAIDS patient serum;

[0039]FIG. 19 is a photograph for immunocytochemistry of BHK-21 cellstransfected with RNA transcript of pSFV/env-gag-pro, showing expressionof Gag, Env and Pro proteins;

[0040]FIG. 20 is a schematic diagram showing a process for constructionof an expression vector pSFV/CTE;

[0041]FIG. 21 is a schematic diagram showing a process for constructionof an expression vector pSFV/gagpro-CTE;

[0042]FIG. 22 is a schematic diagram showing a process for constructionof an expression vector pSFV/env-gag-gagpro-CTE;

[0043]FIG. 23 is a schematic diagram showing a process for constructionof an expression vector pSFV/env-gag-gagΔpro-CTE, showing the insertionof gagΔpro gene, prepared from deletion of a sequence responsible forribosomal frameshifting at 3′ end of gag mRNA region into pSFV/env-gagvector;

[0044]FIG. 24 is a schematic diagram showing a process for constructionof an expression vector pSFV/gagpol;

[0045]FIG. 25 is a photograph for immunocytochemistry showing expressionof Gag and Gagpol polyproteins in BHK-21 cells transfected withpSFV/gagpol vector, with the use of an anti-p24 polyclonal antibody andan anti-protease polyclonal antibody;

[0046]FIG. 26 is a photograph for Western blotting with anti-p24polyclonal antibody in BHK-21 cells transfected with pSFV/gagpol RNAtranscript;

[0047]FIG. 27 is a schematic diagram showing a process for constructionof an expression vector pSFV/envMCTE;

[0048]FIG. 28 is a schematic diagram showing a process for constructionof an expression vector pSFV/gag-envMCTE;

[0049]FIG. 29 is a schematic diagram showing a process for constructionof an expression vector pSFV/gagpol-envMCTE;

[0050]FIG. 30 is a photography for immunocytochemistry showingexpression of Gag and Env proteins in BHK-21 cells transfected withpSFV/gag-envMCTE vector, with the use of an anti-p24 polyclonal antibodyand an anti-protease polyclonal antibody;

[0051]FIG. 31 is a photography for immunocytochemistry showingexpression of Gagpol polyprotein and Env protein in BHK-21 cellstransfected with pSFV/gagpol-envMCTE vector, with the use of an anti-p24polyclonal antibody, an anti-protease polyclonal antibody, and ananti-env monoclonal antibody;

[0052]FIG. 32 is a photograph for Western blotting in BHK-21 cellstransfected with RNA transcripts of pSFV/gag-envMCTE andpSFV/gagpol-envMCTE, with the use of an anti-p24 polyclonal antibody,and an anti-env monoclonal antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0053] The present invention provides alphavirus-based expressionvectors expressing structural proteins of HIV. The terms “HIV” used inthe present invention includes all kinds of human retrovirus such asHIV-1, HIV-2, HTLV-1 and HTLV-2.

[0054] The terms “Alphaviruses” used in the present invention includesvirus species which are categorized into alphavirus and their subtypesin the field of biotechnology and virology such as Eastern EquineEncephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE),Everglades virus Mucambo virus, Pixuna virus, Western Encephalitisvirus(WEE), Sindbis virus, South African Arbovirus No. 86, Girdwood S.A.virus, Ockelbo virus, Semliki Forest virus, Middleburg virus,Chikungunya virus, O'Nyong-Nyong virus, Ross River virus, Barmah Forestvirus, Mayaro virus, Getah virus, Sagiyama virus, Bebaru virus, Mayarovirus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyaylagachvirus, Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creekvirus and other viruses which have been Categorized into alphaviruses bythe International Committee on Taxonomy of Viruses.

[0055] Alphaviruses, which belong to the Togaviridae family, areenveloped positive-strand RNA viruses with a broad range of susceptiblecells including insects, birds, and mammalian animals. An alphavirus RNAgenome is itself infectious and encodes RNA replicase, allowingalphavirus to be used as an expression vector capable of performing RNAreplication and translation (Liljestrom, P. and Graoff, Biotechnology,9, 1356-1361(1991)).

[0056] It is preferable that Alphavirus is selected from “Semliki Forestvirus” (SFV) and “Sindbis virus”, and most preferable, Alphavirus isselected from Semliki Forest virus (SFV). An SFV-based expressionvector, pSFV, provided in an embodiment of the present invention, isprepared from insertion of a SFV cDNA genome into a plasmid harboringSP6 promoter, providing in vitro translation using SP6 polymerase andexpression of foreign genes inserted instead of structural genes, underthe control of 26S subgenomic promoter.

[0057] An in vivo packing system is necessary for introduction ofrecombinant RNA into cells by infection. A pSFV-helper vector contains aRNA replication signal of SFV and genetic information to expressstructural genes, but it lacks a packaging signal for genomic RNA.Accordingly, when the pSFV vector is co-transfected with thepSFV-helper, there can be viral particles produced which carry onlyrecombinant RNA inside of SFV structure proteins. Upon infectingsusceptible cells, the viral particles express recombinant genes, butcannot reconstitute viral particles. With reference to FIG. 2, there isillustrated the structures of pSFV and pSFV-helper vectors used in anembodiment of the present invention. All DNA vectors and their RNAtranscripts utilized in the present invention are within the range ofthe present invention.

[0058] In an aspect of the present invention, there are providedalphavirus-based expression vectors carrying gag, gagpro, gagpol and envgene, respectively, an expression vector carrying env-gag genes, anexpression vector carrying env-gagpro genes, an expression vectorcarrying env-gag-gagpro genes, an expression vector carryingenv-gag-gagΔpro genes and an expression vector carrying gagpol-envgenes, where each gene of HIV is regulated under the alphavirussubgenomic promoter by separately inserting the genes into thedownstream of 26S subgenomic promoter. It is known that expression ofPro synthesis is dependent on the amount of Gag protein (Velissarios K.et al., Virology, 193, 661-672(1993); Magdeleine H. et al., J Virol. 72,4819-4824(1998); Joel G. et al., Virology, 244, 87-96(1998)). To achievemaximum expression of Pro protein through processing of Gag protein,there were constructed expression vectors characterized in that gagΔprogene, in which nucleotides responsible for ribosomal frameshifting aredeleted at the junction between gag and pro genes, or gagpro gene waslinked to 26S promoter, respectively, in addition that env gene locatedat the downstream of gagpol gene was linked to 26S promoter.

[0059] The present invention provides an alphavirus-based expressionvector in which gag gene of HIV-1 is operably linked to a promoter. Asused herein, the term “operably linked” means that the DNA sequencesbeing linked are typically contiguous and in reading frame. In anembodiment of the present invention, a pSFV/gag vector is described(refer to Example 1). The present invention provides an alphavirus-basedexpression vector in which gagpro gene is operably linked to a promoter.A pSFV/gagpro vector is exemplified in an embodiment of the presentinvention (refer to Example 1). Also, the present invention provides analphavirus-based expression vector in which an HIV-1 env gene isoperably linked to a promoter. A pSFV/env vector is exemplified in anembodiment of the present invention (refer to Example 4).

[0060] The present invention provides an alphavirus-based vectorsimultaneously expressing HIV-1 env and gag. More preferably, thepresent invention provides an alphavirus-based expression vectorcomprising the nucleotide sequence of HIV env, operably linked to afirst subgenomic promoter, and the nucleotide sequence of HIV gag,operably linked to the second subgenomic promoter. In an embodiment ofthe present invention, plasmid pSFV/env-gag is exemplified (refer toExample 9). Also, the present invention provides an alphavirus-basedvector comprising the nucleotide sequence of HIV pro, operably linked toa promoter. In an embodiment of the present invention, plasmid pSFV/prois exemplified (refer to Example 9).

[0061] In a further aspect of the present invention, there is providedan alphavirus vector simultaneously expressing env, gag and pro genes ofHIV-1. More preferably, the present invention provides analphavirus-based expression vector comprising env, gag and pro geneswere operably linked to a first, second and third subgenomic promoter,respectively. The vector pSFV/env-gag-pro is exemplified (refer toExample 9).

[0062] The present invention provides an alphavirus vector expressing agagpro gene of HIV-1 and a gene encoding constitutive transport element(CTE), and more preferably an alphavirus-based expression vectorcomprising a gagpro gene operably linked to a promoter in addition to agene encoding CTE. The vector pSFV/gagpro-CTE is exemplified (refer toExample 9).

[0063] Also, the present invention provides an alphavirus vectorexpressing env, gag, gagpro genes of HIV-1 and a gene encoding CTE, andmore preferably an alphavirus-based expression vector comprising env,gag and gagΔpro genes operably linked to a first, second and thirdsubgenomic promoter, respectively, in addition to a gene encoding CTE.The vector pSFV/env-gag-gagpro-CTE is exemplified (refer to Example 12).

[0064] The present invention provides an alphavirus vector expressingenv, gag, and gagΔpro genes of HIV-1 and a gene encoding CTE, and morepreferably an alphavirus-based expression vector comprising env, gag andgagΔpro genes operably linked to a first, second and third subgenomicpromoter, respectively, in addition to a gene encoding CTE. Herein, itshould be noted that the gagΔpro means a gagpro gene prepared fromdeletion of nucleotide sequence at the junctional region between gag andpro genes, providing an open reading frame and allowing expression ofpro protein without frame-shift translation at 3′ end of gag mRNA.Exemplified is pSFV/env-gag-gagΔpro-CTE vector (refer to Example 13).

[0065] The present invention provides an alphavirus vector expressing afull-length gagpro gene of HIV-1, and more preferably analphavirus-based expression vector comprising a full-length gagpro geneoperably linked to a downstream of a subgenomic promoter, with anembodiment of pSFV/gagpol vector (refer to Example 14).

[0066] The present invention provides an alphavirus vectorsimultaneously expressing env and gag genes of HIV-1, and morepreferably an alphavirus-based expression vector comprising env and gaggenes operably linked to a first and second subgenomic promoter,respectively, in addition to a gene encoding MCTE. Exemplified ispSFV/gag-envMCTE vector (refer to Example 16).

[0067] The present invention provides an alphavirus vector,simultaneously expressing gagpol and env genes of HIV-1, and morepreferably an alphavirus-based expression vector comprising gagpol andenv genes operably linked to a first and second subgenomic promoter,respectively, in addition to a gene encoding MCTE. Exemplified ispSFV/gagpol-envMCTE vector (refer to Example 16).

[0068] The expression vectors of the present invention can be prepared,with some modification, using recombinant DNA technology known in thefield to which the present invention belongs.

[0069] The present invention provides a method of preparing HIV-1structural proteins including Gag, Pro and/or Env in the form ofprecursors or processed subunits with the use of alphavirus-basedexpression vectors which are described above. The alphavirus-basedexpression vectors of the present invention can express proteins in awide range of host cells. In addition, the present invention provides amethod of preparing Gag, Pro and/or Env proteins of HIV-1 by expressingthe expression vectors or their RNA transcripts in host cells, and morepreferably, in animal cells. It is preferable that the animal cells areprepared from birds, mammalians, reptiles, amphibians, insects andfishes (aquatic animals), and examples of mammalians include humans,monkeys, hamsters, rats (or mice), and pigs. Most preferably, anexpression system consisting of a host cell and a vector is BHK-21cells/pSFV vector or COS cells/pSFV vector, but the expression system ofthe present invention is not limited to them.

[0070] The alphavirus expression vectors and their RNA transcripts canbe introduced into animal cells, such as BHK-21 cells, by a conventionaltransfection method including DEAE dextran mediated transfection,calcium-phosphate co-precipitation, and more preferably,electroporation. In an embodiment of the present invention, RNAtranscript of pSFV vector was efficiently transfected into a host cellby electroporation. Also, the above transfection methods can be used forco-transfection.

[0071] Expression of foreign genes in animal cells can be maximized bythe introduction of infectious viral particles. Regarding this, thepresent invention provides a method of preparing the above proteins ofHIV, comprising an infection of host cells with an infectious viralparticle harboring RNA transcripts, which is generated using a helpervirus. In Examples 2 and 6, the method is exemplified. HIV-1 proteins,produced by infecting host cells with defective viral particlesgenerated with the use of a helper virus or RNA transcripts of theexpression vectors of the present invention, can be isolated usingconventional methods, including ion-exchange chromatography, affinitychromatography, and gel infiltration chromatography.

[0072] In addition, the present invention provides host cellstransiently transformed with RNA transcripts of the expression vectorsof the present invention and VLP harboring recombinant RNA, and alsoprovides host cells permanently transformed with the expression vectors.The permanently transformed cells can be provided as an animal cell linehaving a cytomegalrovirus (CMV) promoter necessary for integration ofcDNA of HIV genome into chromosome of host cells, a gene encodingconstitutive transport element (CTE) for reversetranscriptase-independent expression, and an anti-neomycin gene as aselective marker. The CTE gene, more preferably, is a gene encoding MPMV(Mason-Pfizer monkey virus).

[0073] The present invention provides a method of preparing aninfectious HIVLP using the expression vectors and the host cellsdescribed above, and more particularly, a method of preparing aGag-containing immature HIVLP by introducing an expression vectorexpressing Gag protein into host cells. When Gag is not processed byprotease, mature viral particles are not generated, resulting inimmature virus-like particles (VLPs). “Virus-like particle (VLP)” meansa virus particle, which is not identical to wild-type (mature) viralparticles, but has similar physicochemical properties to wild types. An“Immature virus-like particle (immature VLP)” is a viral particleshowing the morphology characteristic of viral particles while it isunable to complete the processing necessary for production of wildvirions. “Replicon” means a self-replicating RNA containingindispensable genes for gene replication and is thus capable of beingused as an expression vector for expression of foreign genes while virusproliferates by combination to foreign genes.

[0074] HIV with only Gag protein is capable of producing immature viralparticles without infectivity. Also, it is known that neutralizingantibodies against HIV mainly bind to epitopes located at an envelope(Env) protein that has a variable amino acid sequence. Thus, the Envprotein is a main target for development of HIV vaccines, with growingconcerns for the development of a Gag-containing recombinant viralparticle carrying Env protein. To obtain such a HIV vaccine, the presentinvention provides a method of preparing an immature HIVLP composed ofGag and Env proteins by simultaneously expressing the two proteins inhost cells, which co-transfected with expression vectors harboring gagand env genes.

[0075] On the other hand, the co-transfection method used for theproduction of HIVLP requires many steps and is not effective. Toovercome these problems, the inventors of the present invention used arecombinant plasmid carrying 2 or 3 target genes, offering an economicand effective method in preparing and recovering HIVLP. Accordingly, thepresent invention provides a method of producing an immature HIVLPcomposed of Gag and env proteins, supplied by inserting each of gag andenv genes into the downstream of each promoter of alphavirus repliconand introducing the resulting replicon vector into host cells.

[0076] During an assembly or budding process, or in a viral particle,gag protein is cleaved by protease, and the cleaved subunit proteinsassemble, producing mature particles. The HIVLP composed of only gagprotein varies greatly in size and morphology from a natural HIVLP.Also, unless a HIVLP composed of Gag and Env undergoes the processing byprotease, mature viral particles cannot be produced. To induce aneffective immune response, it is important to provide similar immunogens(antigens) to a wild type HIV. Accordingly, production of mature HIVLPis a strong tool for preparation of a HIV vaccine. In the presentinvention, the inventors developed a method of producing a mature HIVLPthrough the simultaneous expressions of Gag and Pro proteins, or Gag,Env and Pro proteins. The “mature virus-like particle (mature VLP)”referred to (defined) as the HIVLP that has structural proteinscompletely processed by protease (Pro) and shows similar morphology andsize to a wild type HIV. In the present invention, it was found that agreat deal of mature HIVLP was produced when gagpol and env genes weresimultaneously expressed.

[0077] The HIV-like particle (HIVLP) is considered to be a mostpreferable form for HIV vaccines or for antigens for diagnosing of IVinfections. However, expression of Gag, Env, and/or protease in eachdifferent vector to produce HIVLP is complex and ineffective, inaddition that it is very difficult to establish a stable cell lineproducing HIVLP. In the present invention, there was successfullydeveloped an alphavirus-based expression vector simultaneouslyexpressing Gag, Env and protease at amounts large enough to produceHIVLP, as well as successfully prepared and isolated an infectiousHIVLP.

[0078] The present invention further provides a method of preparingenough amount of the above expression vectors for induction of immuneresponse, a vaccine composition including RNA transcripts of theexpression vectors or the HIVLP, and a method of preventing or treatingAIDS comprising administration of the vaccine composition. Preferably,the HIVLP is mature HIVLP, immature HIVLP, infectious HIVLP ornoninfectious HIVLP, and most preferably, mature HIVLP. Due to its threedimensional structure, HIVLP, when it used as a vaccine, inducesexcellent immune response.

[0079] The vaccine composition of the present invention can beadministered to human in a type suitable to living bodies. The typesuitable to living bodies refers to a type of material capable of beingadministered to living bodies with a higher effect of treatment orprevention than its toxic effect. The materials can be administered toanimals, and more preferably to humans.

[0080] The vaccine of the present invention may additionally include asuitable diluent, excipient and/or carrier. The excipient includes asuitable adjuvant capable of improving immunogenicity of the vaccine.The adjuvant can be selected from the group consisting of endotoxinlipid-A of Gram(-) bacteria, trehalos dimycholate of Mycobacteriumspecies, phospholipid lysolecitin, dimethyldioctadecylammonium bromide(DDA), a polyoxypropylene-polyoxyethylene (POP-POE) block copolymer,aluminum hydroxide, and liposome. The carrier may be anything unlessthere are induced unwanted antibodies in administered individuals.Especially, various suitable carriers or diluents such as saline,buffered saline, and a mixture of saline and non-specific serum albuminmay be used for the present invention. The pharmaceutical compositionmay include a vehicle, a buffer, an anti-oxidant, carbohydrate such asglucose, sucrose or dextrin and a chelating agent such as EDTA, andadditionally include water, saline, glycerol, ethanol, an emulsifier, amoistening agent, or a pH adjusting agent.

[0081] The vaccine composition may include an adjuvant capable ofenhancing immune response. Examples of the adjuvant include aluminumhydroxide (alum), thr-MDP, nor-MDP, and MPT-PE. Also, the vaccine mayinclude cytokine such as GM-CSF, IL-2, IL-12, TNF and IFN, which areknown to enhance the immune response.

[0082] The dosage of vaccine may be dependent on factors such as healthcondition, age, sex, body weight and immune response capability ofvaccine. Also, the dosage may vary in order to achieve the maximalresponse. For example, the vaccine may be administrated at a same amountevery day, or the dosage may be gradually reduced in an emergency. Thevaccine may be administered subcutaneously, intravenously,intramuscularly, orally, or intradermally as usual. The inhalation orsuppository administration can be applied. The booster administrationcan be given after 4 to 6 weeks.

[0083] The present invention provides a diagnostic kit for HIV infectionand a diagnostic method using thereof. The diagnostic kit comprises thesteps of: (a) reacting HIVLP with a biological sample from an animal,especially human; and (b) detecting binding of an antibody in the sampleto the HIVLP. Also, there is provided a method of diagnosis of H(Vinfection using the diagnostic kit. Methods to measure the bindingdegree between antigen and antibody are well known in the field to whichthe present invention belongs. ELISA (enzyme-linked immunosorbant assay)is one of the applicable methods. The antigen-antibody binding in thediagnosis can be performed directly or competitively.

[0084] From the viewpoints above, the present invention provides amethod of detection antibodies specific against HIV antigen in the testsample, comprising the steps of: (a) collecting a test sample suspectedto contain a antibodies specific against HIV; (b) reacting the testsample with the mature HIV-like particles prepared according to themethod described in example 18 under a condition which allowsantigen-antibody immune complex to form in the sample; and (c) detectingthe antigen-antibody complexes formed in the test sample.

[0085] The mature HIV-like particles of the present invention, as anantigen, can be useful for performing immunoassay including ELISA, RIAand other non-enzymatically linked antibody tests, or for detectingretrovirus antigens (e.g. HIV antigen) and anti-retrovirus antibodies(e.g. anti-HIV antibody). The mature HIV-like particles, in ELISA, canbe attached onto a specific surface, such as a well of polystyrenemicrotitration plates, where proteins can bind. The plates are washed toremove the unbound mature HIV-like particles. After removingincompletely attached mature HIV-like particles by washing, the plate isblocked with a non-specific protein such as bovine serum albumin (BSA)or casein, reducing non-specific binding of the mature HIVLP to thesurface.

[0086] Thereafter, clinical or biological samples may be applied on thefixed surface, allowing immune complex (antigen-antibody complex)formation. Herein, the sample may be diluted with a diluent such as BSA,bovine gammaglobulin (BGG), or a phosphate buffered solution (PBS)/Tweensolution, followed by incubation at 25° C. and subsequently at 37° C.for 2 to 4 hours. Non-immune complexes are then removed from the surfaceby washing. The washing can be performed by PBS/Tween or boric acidbuffered solutions.

[0087] After washing, a qualitative or qualitative test could beperformed by adding a specific secondary antibody to a first antibody,which formed an immune complex with the mature HIVLP. In the case thatthe sample comes from humans, a secondary antibody is specific againstmainly IgG among human immunoglobulins. To easily detect the immunecomplex, a secondary antibody is conjugated with an enzyme capable ofusing a chromogenic substrate, thus allowing qualitative analysis usinga spectrophotometer.

[0088] To identify multiple antibodies reacting with multiple HIVisolates, multiple HIV-like particles, immunologically different, couldbe attached to a specific surface. To detect an anti-HIV antibodyrecognizing a conserved epitope among IV isolates, one or a limitednumber of mature HIVLP could be attached. To identify antibodiesreacting with one HIV isolate (e.g. LA1, MN, SF2 or HXB2), one matureHIVLP specific to the HIV isolate of the present invention could beattached. This additional diagnostic system can be applied for detectionof a specific TV isolate in clinical tests and medical or medical lawfields.

[0089] In addition, another diagnostic method could be necessary toidentify immunologically different HIV isolates, which belong todifferent classification. Examples of the immunologically different HIVisolates include LA1, MN, SF2, HXB2 and primary HIV-1 isolates. In anaspect of this diagnostic method, the mature HIVLP of the presentinvention could be useful for production of antibodies includingmonoclonal antibodies capable of specifically recognizing theimmunologically different HIV isolates. It is possible that the matureHIVLP of the present invention, which is immunologically different, maybe used as a vaccine and a diagnostic immunogen. A mixture combiningmature HIVLPs could be provided for protection and/or diagnosis ofmixed-isolates. In this case, the mixture of immunogens may be called a“cocktail”.

[0090] A composition having immunogenic properties of the presentinvention, in the non-mixed or cocktail form, can be used for detectingHIV antigen(s) in a sample including a biological sample, or forproducing a HIV-specific antibody (including monoclonal antibody) toneutralize HIV.

[0091] In a further diagnostic application, the mature HIVLP of thepresent invention can be used for stimulating HIV-specific T-cells inbiological samples obtained from HIV-infected patients with an aim fordiagnosis and therapy.

[0092] The present invention will be explained in more detail withreference to the following examples in conjunction with the accompanyingdrawings. However, the following examples are provided only toillustrate the present invention, and the present invention is notlimited to them.

EXAMPLE 1 Construction of Expression Vectors, pSFV/gag and pSFV/gagpro

[0093] Gag protein of HIV and its proteolytic cleavage products are themajor structural components of the virion, and Gag itself is sufficientto direct assembly and release of virus-like particles without any otherviral proteins. Gag protein is cleaved by viral protease (PR) encoded bypol gene, generating matrix (MA, p17), capsid (CA, p24), nucleocapsid(NA, p9), and p6,which are assembled to produce mature viral particles(Gheysen D. et al, Cell, 59, 103-112 (1989)). Therefore, in this study,there was constructed an expression vector capable of expressing Gag byinserting a gag gene into a replicon, as follows. To amplify full-lengtha gag gene, PCR was performed with a primer set consisting of primers 1and 2, below, using HIV lade E genome (Genbank accession No. U51188,Journal of Virologly, 1996), containing full-length gag gene(832 to 2328bp), as a template. Also, to amplify a pro gene in addition to a gaggene, PCR was performed with primers 1 and 3, below, using HIV clade Egenome, containing a gag gene, frame shift site and a pro gene(832 to2577 bp), as a template. In the two cases, a PCR mixture was prepared asfollows: plasmid pUHD (100 ng/μl) carrying full-length HIV-1 clade Egenome, used as the template, was mixed with 4 μl of 2.5 mM dNTP(Boehringer Mannheim(BM), Germany), 1 μl of sense primer (100 pmol/μl),1 μl of antisense primer (100 pmol/μl), 5 μl of 10×Taq polymerasebuffer, 37 μl distilled water. The PCR mixture was pre-incubated at 98°C. for 5 min, and then supplemented with 1 μl of Taq polymerase,followed by a PCR reaction of 30 cycles in which each cycle was composedof 1 min at 94° C., 2 min at 55° C., and 3 min at 72° C. The amplifiedproducts for HIV-1 gag and gagpro were cloned into BamHI site of pSFVvector, respectively, giving recombinant vectors, pSFV/gag (refer toFIG. 3) and pSFV/gagpro (refer to FIG. 4). Primer 1: Sense (SEQ IDNO.: 1) 5′-GCGGATCCCGGGATGGGTGCGAGAGCGTCAATATTAAGT-3′ Primer 2:Antisense (SEQ ID NO.: 2) 5′-CGCGGATCCCTGTTACTGTGACAAGGGGTCGTTGCCAAA-3Primer 3: antisense (SEQ ID NO.: 3)5′-CGCGGATCCCTGCAGTTAAAGTACAACCAATCTGAGTCAACA-3′

EXAMPLE 2 Production of VLP Using pSFV-Helper and pSFV/gag, orpSFV-Helper and pSFV/gagpro

[0094] Protease (Pro) of HIV-1 was activated during or after the buddingof virion, and was involved in the processing of structural polyproteinsincluding Gag and Gagpol precursors. In this example, the recombinantvectors prepared in Example 1, pSFV/gag and pSFV/gagpro, were expressedwith pSFV-helper in host cells.

[0095] pSFV/gag and pSFV/gagpro vectors were completely linearized withthe use of a restriction enzyme PvuI, and purified by phenol/chloroformextraction. A reaction mixture was prepared from 10 μl of the plasmid 10μl of DNA, 5 μl of 10×SP6 buffer (TaKaRa), 2.5 μl of 100 mM DTT, 5 μl ofrNTP mix (10 mM ATP, 10 mM CTP, 10 mM UTP, 5 mM GTP, BM), 2 μl ofRnasin(BM), 1.5 μl of SP6 RNA polymerase (TaKaRa) and 19 μl distilledwater, and then incubated at 37° C. for 1 hour 30 min. Transfection wasthen performed by electroporation, as follows: with 25 μl (1 μg) ofpSFV-helper RNA transcripts, 50 μl of the resulting RNA transcript ofgag or gagpro gene was added to 800 μl of BHK-21 cells (10⁷ cells/ml)suspended in PBS (phosphate buffered saline, 1.37 mM Sodium Chloride,0.02 mM Potassium Chloride, 0.1 mM Phosphate buffer/ml), andelectroporation was then carried out at room temperature in a 0.4 cmelectroporation cuvette using a Bio-Rad Gene Pulser, with two pulses of830 V/25 μF. 48 hours after transfection, the cultivated medium wascentrifuged at 3500 rpm for 15 min and the obtained supernatant was thenultra-centrifuged for 2 hr at 30,000 rpm at 4° C. using a Beckman SW41rotor, giving a pellet containing viral particles. The pellet wasresuspended in TNE buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5 mMEDTA) and stored at −70° C. The defective VLPs contained in the pelletwere eletrophoresed by a SDS-polyacrylamide gel, and were analyzed byWestern blotting using serum from AIDS patients (refer to FIG. 5, laneM: negative control; lane 1: VLP produced from pSFV-helper andpSFV/lacZ; lane 2: VLP produced from pSFV-helper and pSFV/gag; lane 3:VLP produced from pSFV-helper and pSFV/gagpro). In lane 2 of FIG. 5, Gagprotein was detected. This indicates that Gag protein was expressed inBHK-21 cells transfected with pSFV/gag RNA transcripts, and HIVLPscomposed of Gag protein were released into culture medium with SFVLP. Itwas revealed that pSFV/gagpro, prepared for expressing Pro protein aswell as Gag protein produced less Gag protein than pSFV/gag (refer tolane 3 of FIG. 5). BHK-21 cells were infected with the produced viralparticles, as follows: to activate defective VLPs, 100 μl of suspensionof defective VLPs in TNE buffer was incubated with 5 μl of chymotrypsin(10 mg/μl), 5 μl of 50 mM CaCl₂ for 30 min on ice, and 45 μl ofAprotinin (2 mg/μl, Sigma, USA) was then added to terminate the proteaseactivity. The activated VLPs were added to monolayerd BHK-21 cells with70% confluency and then incubated for 90 min at 37° C. under 5% CO₂,allowing for infection of BHK-21 with the VLPs. Culture medium was thenaspirated, and a new medium was refed and incubation was preceded for 48hours. The BHK-21 cells were lysed with 200 μl of a lysis buffer (1%NL40, 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 μg/ml PMSF), andcentrifuged at 12000 rpm for 5 min at 4° C. The supernatant was mixedwith a sample buffer and then boiled for 5 min. The denatured sampleswere separated by SDS-PAGE (12%). The separated proteins weretransferred onto a PVDF membrane (BM) for 2 hrs under a condition of 15V and 7 mA, and Western blotting was performed using specificantibodies, where serum from AIDS patients was used as a first antibodyand biontinylated anti-human IgG as a second antibody. The membrane wasthen reacted with an avidin-biotin solution and the color was developedwith DAB solution (refer to FIG. 6, lane 1: BHK-21 cells infected withVLP from pSFV/gag; lane 2: BHK-21 cells infected with VLP frompSFV/gagpro). As a result of reinfection, the band of 55 kDa presentedthat Gag proteins were produced in cytoplasm of BHK-21 cells. Therefore,these results suggest that VLP obtained from pSFV/gag or pSFV/gagprocontains a gag gene and is infectious.

EXAMPLE 3 Production of VLP Using pSFV/gag or pSFV/gagpro ExpressionVectors

[0096] VLPs prepared in Example 2 are a mixture of VLP composed of acore of SFV (SFVLPgag or SFVLPgagpro) and VLP composed of a core ofHIV-1 (HIVLPgag or SFVLPgagpro). To obtain only VLPs composed of a coreof HIV-1, pSFV/gag and pSFV/gagpro prepared in Example 1 weretranscribed in vitro and transfected into BHK-21 cells withoutpSFV-helper RNA transcripts according to the method of the above Example2. The transfected cells were fixed with cold absolute methanol, cooledat −20° C., for 4 to 6 min, blocked with 1% gelatine, and immunostainedwith a anti-p24 (capsid) polyclonal antibody (anti-rabbit) and then asecondary antibody, biontnylated anti-rabbit IgG. As a result, it wasfound that that Gag protein was expressed in the transfected BHK-21cells (refer to FIG. 7, A: negative control, BHK-21 cells nottransfected; B: BHK-21 cells transfected with pSFV/gag; C: BHK-21 cellstransfected with pSFV/gagpro). Gag proteins were detected in thecytoplasm, and VLPs were isolated from supernatant from the cultivatedmedium by using the method from Example 2, and observed under anelectron microscope (FIG. 8, A: VLPs from pSFV/gag; B: VLPs frompSFV/gagpro). It was found that VLP (HIVLPgag) composed of only Gagproteins was produced even when pSFV/gag or pSFV/gagpro RNA transcriptswere expressed without pSFV-helper RNA transcripts.

EXAMPLE 4 Construction of pSFV/env Expression Vector

[0097] To amplify a gp160 gene of HIV, PCR was performed with a primerset consisting of primers 4 and 5, below, using HIV clade E genome(Genbank accession No. U51188, Journal of Virologly, 1996), containing agp 160 gene between 6264 to 8879 bp, as a template. Primer 4: sense (SEQID NO.: 4) 5′-CGCGCCTCGAGCGGGATCCCATGAGAGTGAAGGGGACACGGA-3′ Primer 5:antisense (SEQ ID NO.: 5) 5′-AATGGATCCTATAGCAAAGCCCTTTCCAAGCCC-3′

[0098] A PCR mixture was prepared as follows: plasmid pUHD (100 ng/μl)carrying full-length HIV-1 clade E genome, used as the template, wasmixed with 4 μl of 2.5 mM dNTP (BM, Germany), 1 μl of sense primer (100pmol/μl), 1 μl of antisense primer (100 pmol/μl), 5 μl of Taq polymerase10× buffer, and 37 μl distilled water. The PCR mixture was pre-incubatedat 98° C. for 5 min, and then supplemented with 1 μl of Taq polymerase,followed by a PCR reaction of 30 cycles in which each cycle was composedof 1 min at 94° C., 2 min at 55° C., and 3 min at 72° C. The amplifiedproduct for gp160 gene was cloned into BamH I site of pSFV vector,giving a recombinant vector, pSFV/env (refer to FIG. 9).

EXAMPLE 5 Production of VLP Using pSFV-Helper and pSFV/env ExpressionVectors

[0099] Because only Env of HIV-1 cannot produce VLP, VLP was preparedfrom pSFV-helper and pSFV/env. The vector pSFV/env prepared in Example 4was completely linearized with SpH I and purified by phenol/chloroformextraction. To obtain RNA transcripts of an env gene, a reaction mixturewas prepared from 10 μl of the linearized plasmid DNA, 5 μl of 10×SP6buffer (TaKaRa), 5 μl of 10 mM m7G(5′)ppp(5′)G(BM), 2.5 μl of 100 mMDTT, 5 μl of rNTP mix (10 mM ATP, 10 mM CTP, 10 mM UTP, 5 mM GTP, BM), 2μl of Rnasin (BM), 1.5 μl of SP6 RNA polymerase (TaKaRa), and 19 μldistilled water, and incubated at 37° C. for 1 hour 30 min. Transfectionwas then performed according to the same electroporation as Example 2,with the exception of the use of 50 μl of pSFV/env RNA transcripts and25 μl of pSFV/helper RNA transcripts as RNA transcripts. 48 hours aftertransfection, the cultivated medium was centrifuged at 3500 rpm for 15min and the supernatant was then ultra-centrifuged(2 hr, 30,000 rpm, 4°C.) using a Beckman SW41 rotor, giving a pellet containing viralparticles. The pellet was resuspended in TNE buffer (50 mM Tris-HCl, pH7.4, 100 mM NaCl, 0.5 mM EDTA) and stored at −70° C. Also, thetransfected cells were fixed with cold absolute methanol, cooled at −20°C., for 4 to 6 min, blocked with 1% gelatine, and immunostained with ananti-gp160 monoclonal antibody (1 mg/ml) and secondarily withbiontnylated anti-mouse IgG (5 μl/ml). The infected cells were thenincubated with an AB solution (5 μl avidin, 5 μlbiotin/1 ml PBS, VECTOR)for 30 min and color was developed with a DAB (3,3′-diaminobezidine)solution (VECTOR) (refer to FIG. 10, A: a negative control,non-transfected BHK cells; B: BHK cells transfected with pSFV/envvector). As a result, it was found that Env protein was expressed in thetransfected BHK-21 cells with pSFV/env vector.

[0100] To activate VLPs, 100 μl of suspension of viral particles in TNEbuffer was incubated with 5 μl of chymotrypsin (10 mg/ml), 5 μl of 5 μlof 50 mM CaCl₂ for 30 min on ice, and 45 μl of Aprotinin (2 mg/ml,Sigma, USA) was then added to terminate the protease activity. Theactivated VLPs were added to monolayerd BHK-21 cells with 70% confluencyand then incubated for 90 min at 37° C. under 5% CO₂, allowing forinfection of BHK-21 with the VLPs. Culture medium was then aspirated,and a new medium was refed and incubation was preceded for 48 hours.After incubation, BHK-21 cells were lysed with 200 μl of a lysis buffer(1% NL40, 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 μg/ml PMSF),and centrifuged(12000 rpm, 5 min, 4° C.). The supernatant was mixed witha sample buffer and then boiled for 5 min. The denatured samples wereseparated by SDS-PAGE (12%). The separated proteins were transferredonto a PVDF membrane (BM) for 2 hrs under a condition of 15 V and 7 mA,and Western blotting was performed using specific antibodies, whereserum from AIDS patients was used as a first antibody and biontinylatedanti-human IgG as a second antibody. The membrane was then reacted withan avidin-biotin solution and the color was developed with DAB solution(refer to FIG. 11, lane 1: a negative control, BHK-21 cells; lane 2:BHK-21 cells transfected with VLP). As a result that BHK-21 cells wereinfected with in vitro activated VLPs, it was found that Env proteinswere produced in cytoplasm of BHK-21 cells, demonstrating production ofinfectious SFVLPenv.

EXAMPLE 6 Production of VLP Using pSFV/gag and pSFV/env ExpressionVectors

[0101] It is well known that Gag protein of HIV directs assembly ofimmature VLP and Env protein of HIV is a major target for development ofneutralizing antibodies (Gheysen D. et al., Cell, 59, 103-112 (1989);Lasky, L. A. et al., Science, 249, 932-935 (1986); Lasky, L. A. et al.,Cell, 50, 975-985 (1987)). Therefore, in order to incorporate Envprotein into viral particles composed of Gag protein, RNA transcripts ofpSFV/gag prepared in Example 1 and pSFV/env prepared in Example 4 wereco-transfected into susceptible host cells. The expression vectors,pSFV/gag and pSFV/env, were transcribed in vitro according to the samemethod as Example 2 (without pSFV-helper), and co-transfected intoBHK-21 cells. At 48 hr after transfection, the transfected cells wereimmunostained with AIDS patient serum and an anti-gp160 monoclonalantibody capable of recognizing Env protein. The sample immunostainedwith the AIDS patient serum (refer to C of FIG. 12) showed much morestaining than that with the anti-gp160 antibody (refer to B of FIG. 12).This result indicates that there was produced HIVLP composed of Gag andEnv proteins of HIV-1(Fig 12 A: negative control; B: anti-gp160monoclonal antibody; C: AIDS patient serum).

EXAMPLE 7 Analysis of HIV-Like Particle-Associated Nucleic Acids

[0102] We investigated whether VLPs prepared in Examples 3 and 6,through transfection with only pSFV/gag RNA transcripts, orco-transfection with both pSFV/gag and pSFV/env RNA transcripts,respectively, carry nucleic acid or not. The VLPs were passed through a10% sucrose cushion. Other RNA and DNA contamination was eliminated by adigestion with 1 mg RNase A and 70 Unit of RNase-free DNase I at roomtemperature for 1 hr in a Mg2SO4-acetate buffer. RNA was purified with aviral RNA purification kit (Viogene), and PCR was then carried out usingthe purified RNA, where there was amplified a mRNA fragment of 650 bp insize as a product for gag, especially p24, and a mRNA fragment of 350 bpas a product for env. For RT-PCR of the purified RNA, the RNA was mixedwith 4 μl of 5 Superscript II reverse transcriptase buffer (250 mMTris-HCl, pH 8.5, 375 mM KCl, 15 mM MgCl₂), 2 μl of 0.1 M DTT and 4 μlof deoxynucleoside triphosphates (dNTP; 25 mM each; TaKaRa) and aprimer, and distilled water up to 20 μl. The mixture was incubated for 2min at 42° C., and supplemented with 1 μl of superscript II reversetranscriptase (from pol gene of Molony Murine Leukemia virus (GibcoBRL)), followed by incubation for 50 min at 42° C. to give first DNAstrands in the form of a RNA/DNA hybrid. Herein, there were used 1 μl ofPrimer 6 of 100 pmol for gag RNA, 1 μl. of Primer 7 of 100 pmol for envRNA. Primer 6: antisense (SEQ ID NO.: 6)5′-CCCAAGCTTTTAGCATGCTGTCATCATTTC-3 Primer 7: antisense (SEQ ID NO.: 7)5′-GGTTCTGCAGAAGCTTCCTTGTTATTTCAAACCA-3

[0103] The RNA/DNA hybrids were denatured and the reverse transcriptasewas inactivated by incubation for 15 min at 70° C. DNA amplification wasdone with Z tag DNA polymerase (TaKaRa) and a primer set (Primer 8 andPrimer 6 for gag RNA detection; Primer 9 and Primer 7 for env RNAdetection) using cDNA made from the above RT-PCR as a template. Herein,10 μl of the template cDNA was mixed with 4 μl of 2.5 mM dNTP (BM,Germany), 1 μl of sense primer (100 pmol/μl), 1 μl of antisense primer(100 pmol/μl), 5 μl of 10× Z Taq polymerase buffer, 28 μl of distilledwater and 1 μl of Z Taq polymerase (TaKaRa). The PCR mixture waspre-incubated for 5 min at 94° C. for denaturation, and then PCR wasperformed with 35 cycles where each cycle consisted of 1 min at 94° C.,1 min at 55° C., and 1 min 72° C., followed by incubation of 7 min at72° C. Primer 8: sense (SEQ ID NO.: 8) 5′-CCCAAGCTTCATCAGGCCTTATCACCT-3Primer 9: sense (SEQ ID NO.: 9) 5′-TCCCCCGGGAAGCTTGCGCAGCAGCATCTGTTG-3

[0104] The amplified products were applied to an agarose gel andvisualized under UV illumination after ethidium bromide staining. It wasfound that a p24 gene was amplified at HIVLP gained after transfectionof only pSFV/gag RNA transcripts as well as HIVLP gained afterco-transfection of both pSFV/gag and pSFV/env RNA transcripts (refer tolanes 1 and 2 in FIG. 13). In contrast, amplication of the gp41 gene wasnot detected at all in the two VLPs (refer to lanes 1 and 3 in FIG. 13).This result suggests that only RNA corresponding to the gag gene waspackaged into VLPs (refer to FIG. 13, lane 1: VLP from pSFV/gag; lanes 2and 3: VLP from co-transfection of pSFV/gag and pSFV/env; lanes 1 and 2were amplified with the use of primers for p24, lane 3 was amplified bythe use of primers for gp41; a 650 bp fragment is a PCR product for gaggene and a 350 bp fragment for the env gene).

EXAMPLE 8 Production of VLP by Infection of Defective SFVLP

[0105] We investigated whether or not VLPs were produced when VLPsobtained from Examples 2 and 5 were in vitro activated and infectedCOS-1 cells. After cell lysate and VLPs were obtained from thesupernatant of a cultivated medium, prepared from the infected COS-1cell with VLP from Example 2 or 5, they were analysed by western-blotwith AIDS patient serum, anti-p24 polyclonal antibody (anti-rabbit) andanti-p24 monoclonal antibody (refer to FIG. 14, A: AIDS patient serum;B: anti-p24 polyclonal antibody; C: anti-p24 monoclonal antibody; anegative control (lanes 1, 4, 7, 10, and 13); infection with SFVLPgag(lanes 2, 5, 8, 11, and 14); co-infection with SFVLPgag and SFVLPenv(lanes 3, 6, 9, 12, and 15); cell lysate (lanes 1, 2, 3, 7, 8, 9, 10,11, and 12); VLP (lanes 4, 5, 6, 13, 14, and 15)).

[0106] As shown in A and B in FIG. 14, when COS-1 cells infected withthe defective SFVLP containing the gag gene, Gag protein was detected inthe cytoplasm of COS-1 cells (lanes 2, and 8) and VLPs from supernatant.In addition, both Gag and Env proteins were detected in the cytoplasm ofCOS-1 cells (lanes 3, and 9) and VLPs from supernatant (lane 6) whenCOS-1 cells co-infected with SFV replicon containing both each gag andenv genes. Accordingly, it was confirmed that, when Gag and Env proteinswere simultaneously expressed in a cell, VLPs composed of the twoproteins were produced. As shown in C of FIG. 14, upon Western blottingwith an anti-p24 monoclonal antibody (Biogenesis, Cat. No. 4999-8607),there was detected a Gag precursor Pr55 and p24 proteins. This explainsthat a Gag precursor could be processed or denatured in COS-1 cellswithout protease of HIV-1.

EXAMPLE 9 Construction of Expression Vectors, pSFV/env-gag andpSFV/env-gag-pro

[0107] To obtain a pro gene (from 2268 to 2606 bp in HIV-1 clade Egenome) containing a 26S subgenomic promoter, PCR was performed with amixture consisting of, pUHD (100 ng/μl) containing whole HIV-1 Clade Egenome, 4 μl of 2.5 mM dNTP (BM, Germany), 1 μl of sense primer (100pmol/μl), 1 μl of antisense primer (100 pmol/μl), 5 μl of 10× Taqpolymerase buffer, 37 μl distilled water, and 1 μl of Taq polymerase,and a thermal cycle of 1 min at 94° C., 1 min at 55° C., and 1 min 72°C. was repeated 40 times. Primer 10: sense (SEQ ID NO.: 10)5′-CCAAGATCTATGACAGCCTCCTCCTTTAGTTTC-3′ Primer 11: antisense (SEQ IDNO.: 11) 5′-CAACCCGGGTCGCGATTAAGTGTCAATAGGACTAAT-3′

[0108] The amplified product of the pro gene was cloned into a SmaI siteof a multicloning site of pSFV using EcoRV and a SmaI recognitionsequence in both ends of the primers, generating pSFV/pro vector (referto FIG. 15). To construct pSFV/env-gag vector, a 26S gag gene wasprimarily amplified using the vector pSFV/gag prepared in Example 1 as atemplate, in the presence of Primer 12 (sense) for 26S subgenomicpromoter and Primer 2 (antisense) for a gag gene. The amplified 26S gaggene was then inserted into the SmaI site of the vector pSFV/env (referto FIG. 16). Primer 12: sense (SEQ ID NO.: 12)5′-CCGGATATCACCTCTACGGCGGTCCTA-3′ Primer 13: antisense (SEQ ID NO.: 13)5′-CGCCCGGGTTACTGTGACAAGGGGTCGTTGCCAAA-3′

[0109] Using the pSFV/pro vector as a template, a 26S pro gene wasamplified with a primer set of Primer 12 (sense) for a 26S subgenomicpromoter and Primer 11 (antisense) for pro gene, and the amplifiedproduct was then inserted into the SmaI site of a pSFV/env-gag vector,giving a pSFV/env-gag-pro vector (refer to FIG. 17). Herein, thenucleotide sequence of the amplified pro gene is in part deleted atN-terminus and gained at C-terminus, in comparison with a pro gene ofgagpro in Example 1. Thus obtained sequence for a pro gene contains anucleotide sequence necessary for self-processing (Viviane V et al., J.Gen. Virol. 73, 439-651 (1992)). The vector pSFV/env-gag-pro wasdeposited with Korean Culture Center of Microorganisms(KCCM), one ofinternational depository authorities, on December 2000, as Accession NO.KCCM-10233, under the Budapest Threaty on the international Recognigionof the Deposit of Microorganisms for the purpose of Patent Procedure.

EXAMPLE 10 Production of VLP Through Expression of pSFV/env-gag Vector

[0110] To produce gag-containing VLPs into which Env protein isincorporated, the Gag and Env protein genes were cloned into a SemlikiForest Virus (SFV) replicon in the same plasmid with separate subgenomicpromoters, as described in Example 9. RNA transcripts of the resultingpSFV/env-gag vector of Example 9 were transfected into BHK-21 cells. At48 hr after transfection, the transfected cell lysates and VLPs obtainedfrom the supernatant were analyzed by Western blot with AIDS patientserum, followed by ECL (enhanced chemiluminescence) assay (refer to FIG.18, lane 1: negative control; lanes 2, and 3: cell lysate; lanes 4, and5: cell supernatant). As a result, both Gag and Env proteins weredetected in VLPs, demonstrating env and gag genes cloned on the sameplasmid, in which the expression of the two genes were separatelycontrolled under different subgenomic promoters, were simultaneouslyexpressed in the same cell, allowing production of VLP by theinteraction of the two proteins. In FIG. 18, in the supernatant of acultivated medium, band for Gag protein was shown in a slightly smallersize than in cell lysate because of being pushed by a high content ofalbumin of 65 kDa.

EXAMPLE 11 Production of VLP Through Expression of pSFV/env-gag-proVector

[0111] The pSFV/env-gag-pro vector prepared in Example 9 was transfectedinto host cells and expression levels for the three genes were examined.RNA transcripts of pSFV/env-gag-pro were transfected into BHK-21 cells.At 48 hr after transfection, the transfected cells were immunostainedwith AIDS patient serum, anti-gp160 monoclonal antibody, anti-p24polyclonal antibody, and anti-protease polyclonal antibody (anti-sheep)(refer to FIG. 19, A: a negative control; B: AIDS patient serum; C:anti-gp160 monoclonal antibody; D: anti-p24 polygonal antibody; E:anti-protease polyclonal antibody). As a result, Gag, Env, and Proproteins were all detected. Accordingly, it indicates that Gag, Env, andPro were separately expressed under different 26S subgenomic promoters,demonstrating that there can be expressed each of three foreign genes,separately linked to a subgenomic promoter of an alphavirus-basedexpression vector

EXAMPLE 12 Construction of pSFV/env-gag-gagpro-CTE Expression Vector

[0112] To primarily make a pSFV/CTE vector, PCR was performed withPrimers 14 and 15 using pGEM 7fz(-)/MPMV plasmid DNA as a template. Theamplified CTE gene was inserted into a region between a BamHI site and aSmaI site of pSFV (refer to FIG. 20). A Gagpro gene excised frompSFV/gagpro, prepared in Example 1 by digestion with BamHI, was clonedinto a BamHI site of the vector pSFV/CTE, giving pSFV/gagpro-CTE (referto FIG. 21). A CTE gene from Maston-Pfzier monkey virus was theninserted behind a pSFV/gagpro, generating pSFV/gagpro-CTE vector.

[0113] To finally construct pSFV/env-gag-gagpro-CTE, using the vectorpSFV/gagpro-CTE as a template, a full-length gagpro gene containing 26Ssubgenomic promoter and a CTE gene were amplified by PCR with primers 12and 15. The resulting product was inserted into a SmaI site of thevector pSFV/env-gag of Example 9 (refer to FIG. 22). A PCR mixture wasprepared as follows: 1 μl of pSFV/gagpro-CTE was mixed with 4 μl of 2.5mM dNTP (BM, Germany), 1 μl of sense primer (100 pmol/μl), 1 μl ofantisense primer (100 pmol/μl), 5 μl of 10× Taq polymerase buffer and 37μl distilled water. The PCR mixture was pre-incubated at 98° C. for 5min, and then supplemented with 1 μl of Taq polymerase, followed by PCRreaction of 40 cycles in which each cycle consisted of 1 min at 94° C.,1 min at 55° C., and 2 min at 72° C. The vector pSFV/env-gag-gagpro-CTEwas deposited with Korean Culture Center of Microorganisms(KCCM), one ofinternational depository authorities, on December 2000, as Accession No.KCCM-10234, under the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure.Primer 14 sense (SEQ ID NO.: 14) 5′-AATGGATCCCCTCCCCTGTGAGCTAGACT-3′Primer 15 antisense (SEQ ID NO.: 15)5′-AATGATATCAGATCTCCAAGACATCATCCGGGCAA-3′

EXAMPLE 13 Construction of pSFV/env-gag-gagΔpro-CTE Expression Vector

[0114] gagΔpro was prepared from deletion of conserved five Ts at asignal for ribosomal frame shift from a gagpro sequence, as follows.Using the vector pSFV/gag of Example 1 as a template, amplified was aportion containing 26S subgenomic promoter and gag gene (832-2113),located upstream of site, with the use of Primer 12 and Primer 16,below. Herein, a PCR mixture was prepared as follows: 1 μl of pSFV/gagwas mixed with 4 μl of 2.5 mM dNTP (BM, Germany), 1 μl of sense primer(100 pmol/μl), 1 μl of antisense primer (100 pmol/μl), 5 μl of 10× Taqpolymerase buffer and 37 μl distilled water. The PCR mixture waspre-incubated at 98° C. for 5 min, and then supplemented with 1 μl ofTaq polymerase, followed by a PCR reaction of 40 cycles in which eachcycle consisted of 1 min at 94° C., 1 min at 55° C., and 1 min at 72° C.The amplified product was inserted into pGEMT vector (Promega). Also, afull-length pro gene, with the exception of the conserved seqeuce of asignal for frameshifting, was amplified using the vector pSFF/gagpro-CTEof Example 12 as a template with a primer set of Primer 15 and 17, andthen inserted into pGEMT vector, according to the same proceduredescribed above. Primer 16 antisense (SEQ ID NO.: 16)5′-AATAGGCCTGTCTTTCAGTGCAGTCTT-3′ Primer 17 sense (SEQ ID NO.: 17)5′-CACAGGCCTATAGGGAAAATCTGGCCTTC-3′

[0115] The two obtained genes were digested with restriction enzyme StuIand ligated, generating gagΔpro. In this step, Asn (Asparagine) wassubstituted with Tyr (Tyrosine), and two Phe(Phenylalanine) deleted withthe deletion of conserved five Ts. gagΔpro was then digested with EcoRVand inserted into a SmaI site of the vector pSFV/env-gag, givingpSFV/env-gag-gagΔpro-CTE vector (refer to FIG. 23). (SEQ ID NO.: 18) F  F  R  E CAG GCT AATTT TTT AGG GAA Ribosomal frame shift site ofgag-pol mRNA     Q    A    N (SEQ ID NO.: 19) CAG GCC TAT AGG GAAMutation site of GagΔpro  Q   A   Y   R   E

EXAMPLE 14 Construction of pSFV/gagpol Expression Vector

[0116] To prepare an expression vector expressing a Gagpol polyprotein,a gagpol gene of HIV-1 was inserted into an alphavirus replicon. Afull-length gagpol gene (832-5132 bp) of HIV-1 was amplified bypolymerase chain reaction (PCR) with Primers 18 and 19 using HIV-1 CladeE genome as a template. A PCR mixture was prepared as follows: plasmidpUHD (100 ng/μl) carrying full-length HIV-1 clade E genome, used as thetemplate, was mixed with 4 μl of 2.5 mM dNTP (BM, Germany), 1 μl ofsense primer (100 pmol/μl), 1 μl of antisense primer (100 pmol/μl), 5 μlof 10× Taq polymerase buffer and 37 μl distilled water. The PCR mixturewas pre-incubated at 98° C. for 5 min. and then supplemented with 1 μlof Taq polymerase, followed by a PCR reaction of 40 cycles in which eachcycle consisted of 1 min at 94° C., 1 min at 55° C., and 3 min at 72° C.Primer 18: sense (SEQ ID NO.: 20) 5′-TTAGGATCCATGGGTGCGAGAGCGTCA-3′Primer 19: antisense (SEQ ID NO.: 21)5′-CGCGGATCCCTAATCCTCATTCTGTCTACC-3′

[0117] The amplified product was inserted into a BamHI site of pSFVvector, giving a pSFV/gagpol vector (refer to FIG. 24).

EXAMPLE 15 Preparation of VLP Using pSFV/gagpol Vector

[0118] To produce VLP composed of processed HIV-1core proteins, thevector pSFV/gagpol prepared in Example 14 was in vitro transcribed andtransfected into BHK-21 cells without pSFV-helper RNA transcriptsaccording to the same method as Example 2. The transfected cells werefixed with cold absolute methanol, cooled at −20° C., for 4 to 6 min,blocked with 1% gelatine, and immunostained with AIDS patient serum,anti-p24 polyclonal antibody and anti-pro polyclonal antibody (refer toFIG. 25, A: a negative control; B: AIDS patient serum; C: anti-p24polyclonal antibody; D: anti-pro polyclonal antibody). It was found thatGagpol polyprotein as well as Gag protein was expressed in thetransfected cells. BHK-21 cells were transfected with pSFV/gagpol RNAtranscripts, after an incubation period of 48 hr, the transfected celllysates and VLPs from the supernatant were analyzed by Western blottingwith an anti-p24 polyclonal antibody, followed by ECL assay (refer toFIG. 26, lanes 1 and 4: a negative control; lanes 2 and 5: sample frompSFV/gag; lanes 3 and 6: sample from pSFV/gagpol; cell lysate (lanes 1,2, and 3); VLP (lanes 4, 5, and 6)).

[0119] As shown in FIG. 26, BHK-21 cells transfected with pSFV/gagvector expressed only Pr55 and released immature VLPs. In contrast,BHK-21 cells transfected with pSFV/gagpol vector expressed p24 inaddition to Pr55, and released processed mature VLPs, as a resultprocessed Pr55 positively regulated the expression of p24.

EXAMPLE 16 Construction of pSFV/gag-envMCTE and pSFV/gagpol-envMCTEExpression Vectors

[0120] To produce mature VLP carrying Env protein, an env genecontaining 26S subgenomic promoter and MCTE was amplified and insertedinto a SmaI site of the vector pSFV/gagpol, giving pSFV/gagpol-envMCTEvector. Also, as a comparative construct, pSFV/gag-envMCTE vector wasprepared. To construct pSFV/gag-envMCTE vector, primarily, pSFV/gagvector was newly prepared. Because the vector pSFV/gag made in Example 1contains a SmaI site in front of the gag gene, a full-length gag gene(832-2328 bp) of HIV-1 was again amplified by PCR using Primer 18without SmaI site and Primer 2 according to the same method asExample 1. Next, pSFV/envMCTE (refer to FIG. 27) was prepared byinserting an env gene at a BamHI site of pSFV/MCTE according to the samemethod as Example 4. 26SenvMCTE was amplified by using pSFV/envMCTE as atemplate with Primer 12 (sense) for 26S subgenomic promoter and Primer15 (antisense) for MCTE, and inserted into a SmaI site of pSFV/gagvector, finally giving pSFV/gag-envMCTE (refer to FIG. 28). Also, theamplified 26SenvMCTE gene was inserted into SmaI site of the vectorpSFV/gagpol, giving pSFV/gagpol-envMCTE vector (refer to FIG. 29). Thevector pSFV/gagpol-env-MCTE was deposited with Korean Culture Center ofMicroorganisms, one of international depository authorities, on December2001, as Accession No. KCCM-10348, under the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

EXAMPLE 17 Production of VLP Using pSFV/gag-envMCTE andpSFV/gagpol-envMCTE Expression Vectors

[0121] pSFV/gag-envMCTE was in vitro transcribed, and the transcript wastransfected into the BHK-21 cells without pSFV-helper RNA transcriptsaccording to the same method as Example 2. The transfected cells werefixed with cold absolute methanol, cooled at −20° C., for 4 to 6 min,blocked with 1% gelatine, and immunostained with AIDS patient serum,anti-p24 polyclonal antibody, anti-pro polyclonal antibody and anti-envmonoclonal antibody (refer to FIG. 30, A: a negative control; B: AIDSpatient serum; C: anti-p24 polyclonal antibody; D: anti-env polyclonalantibody). It was found that Env protein as well as Gag protein wasexpressed in the transfected cells.

[0122] Also, a pSFV/gagpol-envMCTE vector was transfected using the samemethod as Example 2 (refer to FIG. 31, A: a negative control; B: AIDSpatient serum; C: anti-p24 polyclonal antibody; D: anti-pro polyclonalantibody; E; anti-env monoclonal antibody). It was also reveled that Envprotein as well as Gagpol protein was expressed in the transfectedcells. In addition, at 48 hr after transfection of the RNA transcriptsof pSFV/gag-envMCTE and pSFV/gagpol-envMCTE into the BHK-21 cells, VLPsisolated from supernatant of the cultivated medium were analyzed byWestern blotting with anti-p24 polyclonal antibody and anti-envmonoclonal antibody, followed by ECL assay (refer to FIG. 32, A: AIDSpatient serum; B: anti-env monoclonal antibody; lanes 1, and 4: samplefrom pSFV/gag; lanes 2, and 5: sample from pSFV/gag-envMCTE; lanes 3,and 6: sample from pSFV/gagpol-envMCTE). As a result, it was discoveredthat, when pSFV/gagpol-envMCTE expressed, there were produced processedmature VLPs carrying Env

1 21 1 39 DNA Artificial Sequence Primer 1 gcggatcccg ggatgggtgcgagagcgtca atattaagt 39 2 39 DNA Artificial Sequence Primer 2 cgcggatccctgttactgtg acaaggggtc gttgccaaa 39 3 42 DNA Artificial Sequence Primer 3cgcggatccc tgcagttaaa gtacaaccaa tctgagtcaa ca 42 4 42 DNA ArtificialSequence Primer 4 cgcgcctcga gcgggatccc atgagagtga aggggacacg ga 42 5 33DNA Artificial Sequence Primer 5 aatggatcct atagcaaagc cctttccaag ccc 336 30 DNA Artificial Sequence Primer 6 cccaagcttt tagcatgctg tcatcatttc30 7 34 DNA Artificial Sequence Primer 7 ggttctgcag aagcttccttgttatttcaa acca 34 8 27 DNA Artificial Sequence Primer 8 cccaagcttcatcaggcctt atcacct 27 9 33 DNA Artificial Sequence Primer 9 tcccccgggaagcttgcgca gcagcatctg ttg 33 10 33 DNA Artificial Sequence Primer 10ccaagatcta tgacagcctc ctcctttagt ttc 33 11 36 DNA Artificial SequencePrimer 11 caacccgggt cgcgattaag tgtcaatagg actaat 36 12 27 DNAArtificial Sequence Primer 12 ccggatatca cctctacggc ggtccta 27 13 35 DNAArtificial Sequence Primer 13 cgcccgggtt actgtgacaa ggggtcgttg ccaaa 3514 29 DNA Artificial Sequence Primer 14 aatggatccc ctcccctgtg agctagact29 15 35 DNA Artificial Sequence Primer 15 aatgatatca gatctccaagacatcatccg ggcaa 35 16 27 DNA Artificial Sequence Primer 16 aataggcctgtctttcagtg cagtctt 27 17 29 DNA Artificial Sequence Primer 17 cacaggcctatagggaaaat ctggccttc 29 18 20 DNA Artificial Sequence Frame shift partof Gag-pol 18 caggctaatt ttttagggaa 20 19 15 DNA Artificial SequencePart of HIV genomic DNA 19 caggcctata gggaa 15 20 27 DNA ArtificialSequence Primer 20 ttaggatcca tgggtgcgag agcgtca 27 21 30 DNA ArtificialSequence Primer 21 cgcggatccc taatcctcat tctgtctacc 30

What is claimed is:
 1. An alphavirus-based expression vector comprisingthe nucleotide sequence (6264-8879 bp) of HIV env, operably linked to afirst subgenomic promoter, and the nucleotide sequence (832-2328 bp) ofHIV gag, operably linked to a second subgenomic promoter.
 2. Theexpression vector as set forth in claim 1, which is plasmidpSFV/env-gag.
 3. An alphavirus-based expression vector comprising thenucleotide sequence (832-2328 bp) of HIV gag, operably linked to a firstsubgenomic promoter, and the nucleotide sequence (6264-8879 bp) of HIVenv, operably linked to a second subgenomic promoter.
 4. The expressionvector as set forth in claim 3, which is plasmid pSFV/gag-env.
 5. Analphavirus-based expression vector comprising the nucleotide sequence(6264-8879 bp) of HIV env, operably linked to a first subgenomicpromoter, the nucleotide sequence (832-2328 bp) of HIV gag, operablylinked to a second subgenomic promoter, and the nucleotide sequence(2268-2606 bp) of HIV pro, operably linked to a third subgenomicpromoter.
 6. The expression vector as set forth in claim 5, which isplasmid pSFV/env-gag-pro.
 7. An alphavirus-based expression vectorcomprising the nucleotide sequence (6264-8879 bp) of HIV env, operablylinked to a first subgenomic promoter, the nucleotide sequence (832-2328bp) of HIV gag, operably linked to a second subgenomic promoter, and thenucleotide sequence (832-2577 bp) of HIV gagpro, operably linked to athird subgenomic promoter.
 8. The expression vector as set forth inclaim 7, which is plasmid pSFV/env-gag-gagpro.
 9. The expression vectoras set forth in claim 7, which is plasmid pSFV/env-gag-gagΔpro.
 10. Analphavirus-based expression vector comprising the nucleotide sequence(832-5132 bp) of HIV gagpol, operably linked to a first subgenomicpromoter, and the nucleotide sequence (6264-8879 bp) of HIV env,operably linked to a second subgenomic promoter.
 11. The expressionvector as set forth in claim 10, which is plasmid pSFV/gagpol-env. 12.The expression vector as set forth in any one of claims 1 to 11, whichfurther comprises a constitutive transport element (CTE).
 13. Theexpression vector as set forth in claim 12, which is plasmidpSFV/env-gag-gagpro-CTE.
 14. The expression vector as set forth in claim12, which is plasmid pSFV/env-gag-gagΔpro-CTE.
 15. The expression vectoras set forth in claim 12, which is plasmid pSFV/gagpol-env-MCTE
 16. Theexpression vector as set forth in claim 12, which is plasmidpSFV/gag-env-MCTE.
 17. An expression vector deposited in the KoreanCulture Center of Microorganisms (KCCM) with accession No. KCCM-10233.18. An expression vector deposited in the Korean Culture Center ofMicroorganisms (KCCM) with accession No. KCCM-10234.
 19. An expressionvector deposited in the Korean Culture Center of Microorganisms (KCCM)with accession No. KCCM-10348.
 20. A method of preparing HIV-likeparticles, comprising the steps of introducing the expression vector ofany one of claims 1 to 19 or its RNA transcript into animal cells, andallowing the expression vector and the RNA to produce HIV-likeparticles.
 21. The method as set forth in claim 20, wherein the HIV-likeparticles are mature HIV-like particles.
 22. The method as set forth inclaim 21, wherein the mature HIV-like particles are infectious.
 23. Ahost cell transformed with one selected from the expression vectors ofany one of claims 1 to
 19. 24. The host cell as set forth in claim 23,wherein the host cell is stably transformed with the expression vector.25. An AIDS vaccine composition comprising a sufficient amount of theexpression vectors of any one of claims 1 to 19 or its RNA to induceimmune response.
 26. An AIDS vaccine composition comprising a sufficientamount of mature HIV-like particles prepared according to the method ofclaim 21 to induce immune response.
 27. A method of vaccinating humansagainst AIDS comprising administration of the AIDS vaccine compositionof claim 25 or 26 to the humans.
 28. A diagnostic kit for HIV infectioncomprising the mature HIV-like particles prepared according to themethod of claim
 21. 29. A method of detecting antibodies specificagainst HIV antigens in the test sample, comprising the steps of: (a)collecting a test sample suspected to containing antibodies specificagainst HIV; (b) reacting the test sample with the mature HIV-likeparticles prepared according to the method of claim 21 under a conditionwhich allows antigen-antibody immune complex to form in the sample; and(c) detecting the antigen-antibody complexes formed in the test sample.